Non Technical Summary
Farmers use fertilizers to provide corn with the essential element, nitrogen. However, current nitrogen fertilizer strategies are inefficient and negatively impact the environment. Non-renewable and polluting natural gas is required to generate most nitrogen fertilizers. Additionally, the process of applying nitrogen fertilizers to fields is inefficient; greater than half of the nutrients can be lost to agricultural runoff instead of supporting crop growth. This also causes environmental damage like algae blooms in lakes and contamination of drinking water. As an alternative, scientists are interested in using microbes that live in the soil and on corn roots to provide crops with nitrogen. Diazotrophs are a type of microbes that can convert nitrogen in the air into a form accessible to plants through a process called nitrogen fixation. While researchers have extensively studied individual diazotrophs, less is known about how diazotrophs interact with each other and the other microbes that live around corn roots.In my research project, I am interested in studying interactions within the community of microbes living around corn roots and how these interactions impact nitrogen fixation. I will use this information to design microbial mixtures that could be applied to crops as fertilizer alternatives. I plan to do this by combining different species in the lab and observing how they grow together. I will additionally test the total nitrogen fixation activity produced by these different microbial combinations. Then, I will use these data to build mathematical models to predict which species combinations will lead to improved nitrogen fixation activity. Using similar approaches, I will further investigate how different nutrients secreted by corn roots impact community growth and nitrogen fixation. Nitrogen fixation can be inhibited by the presence of oxygen. I therefore will engineer key microbial strains to further support nitrogen fixation through oxygen consumption. Through this research project I will generate microbially-based fertilizer alternatives for corn as well as provide the agricultural research field with further insight into the process of nitrogen fixation.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
401	4010	1103	100%

Knowledge Area
401 - Structures, Facilities, and General Purpose Farm Supplies;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1103 - Other microbiology;

Goals / Objectives
In the face of the twin challenges of climate change and an increasing world population, there is a need for improved sustainability in the agricultural industry. In particular, cultivation of the major cereal crop maize, relies heavily on natural gas intensive and environmentally damaging synthetic nitrogenous fertilizers. Using nitrogen fixing microbes as biofertilizers has the potential to improve the sustainability of maize cultivation, however knowledge of the bacterial interspecies interactions governing nitrogen fixation in the rhizosphere microbiome remains limited. I propose to investigate the ecological and molecular mechanisms affecting nitrogen fixation in a synthetic microbiome system and use this knowledge to develop microbial consortia for use as robust crop inoculants. I will use high-throughput in vitro experimentation paired with computational modeling to elucidate the dynamics of community assembly and nitrogen fixing ability. I further propose to determine how the interspecies interactions present within the maize rhizosphere microbiome are affected by changes in the root exudate nutrient environment. Additionally, I will explore the effect of oxygen levels on community nitrogen fixation, metabolically engineering key, non-diazotrophic species for enhanced oxygen consumption. Through this interdisciplinary postdoctoral research project, I will provide the agricultural field with insight into the workings of the rhizosphere microbiome as well as designed microbial communities for use in sustainable fertilizers strategies.1. Design microbial community inoculants for enhanced nitrogen fixation.2. Design communities robust to changes in root exudation.3. Engineer key, non-diazotrophic strains to support community nitrogen fixation through oxygen consumption.

Project Methods
In order to investigate the community properties governing diazotroph fixing and growth in a high-throughput and quantitative manner, I propose to use a synthetic microbial community approach. In contrast to a natural community, using a synthetic community will allow me to control the initial abundance and presence/absence of each species. I drew upon a previous study which identified a seven-member community from maize roots and added five additional diazotrophs to create a 12-member synthetic community I refer to as PComm1.Aim 1.1: Investigate the ecological interactions shaping community nitrogen fixation. I hypothesize that interspecies interactions support nitrogen fixation in the maize rhizosphere microbiome and propose to resolve these interactions by measuring the growth and nitrogen fixation of subcommunities of PComm1. Acetylene reduction assays provide a valuable method for probing nitrogenase activity directly however, this technique is not amendable to high-throughput experimentation. I therefore plan to evaluate ammonia production of each diazotroph alone as well as that of the full 12-member community, in a time-course experiment and to compare these results to those obtained through acetylene reduction assays. Using multiplexed 16S rRNA gene sequencing to evaluate species abundance, I will culture all possible one, two, 11 and 12-member communities in artificial root exudate (ARE) media under oxygen limited conditions. The resulting compositional and functional dataset will allow the identification of interspecies interactions that positively or negatively impact ammonia production. Additionally, this data will be used for model parameterization in Aim 1.2.Aim 1.2: Develop a computational model to predict and design community-level nitrogen fixation. I propose to use a dynamical systems and statistical modeling hybrid approach to further understand this complex system as well as guide the design of subcommunities for use as microbial inoculants. In order to quantitatively examine community growth, I will use the generalized Lotka-Volterra (gLV) model, which is commonly used in microbial ecology because in contrast to other modelling approaches, the gLV model provides an interpretable model form where estimated interaction parameters are biologically relevant. I will estimate the gLV model parameters from the monospecies and pairs absolute abundance data obtained in Aim 1.1 using a Bayesian parameter inference approach. In addition to modeling growth dynamics, I plan to examine community function using linear regression. Using this two-stage modelling approach, I will simulate the growth and ammonia production from untested subcommunities of PComm1.Aim 1.3: Develop and test microbial communities optimized for nitrogen fixation. Guided by the computational models parametrized in Aim 1.2, I will select a set of approximately 50 communities for in vitro validation. Using the high-throughput experimental conditions refined in Aim 1.1, I will evaluate community composition through 16S sequencing and community function through ammonia colorimetric assays. By comparing the resulting data to the simulated data obtained in Aim 1.2, I will evaluate the predictive power of my model and identify communities of interest for low-throughput assays on maize seedlings. I will select the top five ammonia producing communities for further evaluation. These communities will then be inoculated onto maize seedlings and nitrogenase activity will be assessed via acetylene reduction assays. As a control I will compare the in planta nitrogenase activity of the designed communities to that of individual diazotrophic strains. Aim 2.1: Explore exudate metabolite space through media design. Nutrient availability and resource competition are major forces that drive microbial community assembly and function. Understanding how microbial communities respond to variation in the rhizosphere environment would enable identification of key inputs to control nitrogen fixation. I therefore propose to explore the maize root exudate metabolite space by designing a suite of media that vary the presence/absence and concentration of metabolites found in exudates. My media design strategy will include sugars, organic acids and amino acids, in order to explore the different carbon and nitrogen sources available to microbial communities. I will use the statistical software, JMPâ Pro, to computationally identify metabolite combinations that effectively sample the design space and maximize the information my experiments will be able to glean.Aim 2.2: Computationally design communities robust to environmental perturbations. I therefore to investigate ammonia production across a range of exudate profiles using a small set of communities, including the full 12-member community, PComm1, as well as subcommunities of interest identified in Aim 1.3. Using the suite of media developed in Aim 2.1, I will investigate the effect of these varied media conditions on biological nitrogen fixation using a colorimetric ammonia assay and 16S rRNA gene sequencing. I will use the resulting data to train an input-output model to predict ammonia production as a function of the various media and species inputs. I will design communities predicted to have consistent ammonia production across media inputs. I will then test the ammonia production ability of these communities in vitro using the suite of media developed in Aim 2.1.Aim 3.1: Engineer Enterobacter cloacae and Pseudomonas putida for increased oxygen consumption. I hypothesize that engineering non-diazotrophic community members for enhanced oxygen consumption may further support community nitrogen fixation. Previous research has demonstrated that the activity of the electron transport chain and thus oxygen consumption, can be modulated via ubiquinone availability. I therefore plan to metabolically engineer increased flux towards ubiquinone synthesis via overexpression of the key branch point gene, UbiC, as well as upstream deregulation of the Shikimate pathway as demonstrated in E. coli. As an alternative method, I plan to decrease the ratio of ATP produced to oxygen consumed, using low efficiency energy generation to drive oxygen consumption. I plan to drive electron flux to less efficiency energy generation via overexpression of the native cytochrome bd and heterologous expression of the Azotoboacter vinelandii NADH:ubiquinone oxidoreductase. I will evaluate the ability of these engineered strains to promote fixing via co-culture with Klebsiella variicola, as described in Aim 3.1.Aim 3.2: Evaluate community response to engineered strains. Following the metabolic engineering of oxygen consuming strains in Aim 3.2, I plan to assess how these engineered strains effect community growth and nitrogen fixation activity. I will substitute engineered strains for wildtype Enterobacter cloacae or Pseudomonas putida in PComm1 as well as communities of interest developed in Aims 1 and 2, creating several synthetic communities containing one metabolically engineered strain each. I will perform high-throughput community experiments under ambient oxygen levels, using 16S sequencing and ammonia colorimetric assays to determine community composition and function. I will perform these experiments in 96-well plates equipped with an oxygen sensor in each well, enabling regular oxygen measurements throughout the experiment using a fluorescent microplate reader. This data will provide insight into the effect of increased oxygen consumption on previously observed community dynamics. Additionally, I will identify ammonia producing communities for in planta evaluation of nitrogenase activity via acetylene reduction assays. This experiment will allow me to determine the ability of these engineered strains to promote nitrogenase activity in the presence of the oxygen gradient found on the maize root.